The present invention relates to expression of recombinant proteins in eukaryotic cells.
The development of expression systems for production of recombinant proteins is important for providing a source of a given protein for research or therapeutic use. Expression systems have been developed for both prokaryotic cells, such as E. coli, and for eukaryotic cells, such as yeast (i.e., Saccharomyces, Pichia and Kluyveromyces spp) and mammalian cells. Expression in mammalian cells is often preferred for manufacturing of therapeutic proteins, since post-translational modifications in such expression systems are more likely to resemble those occurring on endogenous proteins in a mammal, than the type of post-translational modifications that occur in microbial expression systems.
Several vectors are available for expression in mammalian hosts, each containing various combinations of cis- and in some cases trans-regulatory elements to achieve high levels of recombinant protein in a minimal time frame. However, despite the availability of numerous such vectors, the level of expression of a recombinant protein achieved in mammalian systems is often lower than that obtained with a microbial expression system. Additionally, because only a small percentage of cloned, transfected mammalian cells express high levels of the protein of interest, it can often take a considerably longer time to develop useful stably transfected mammalian cell lines than it takes for microbial systems.
The use of a dicistronic expression vector wherein a first open reading frame encodes a polypeptide of interest and a second open reading frame encodes a selectable marker, is one method that has been used to obtain recombinant proteins. A preferred marker for use in such systems is dihydrofolate reductase (DHFR), which has the advantage of being an amplifiable gene, allowing selection for cells having high copy numbers of the inserted DNA by culturing them in increasing levels of methotrexate (MTX). However, translation of the selectable marker gene is up to 100-fold less efficient than translation of the gene of interest, which reduces the efficiency of the selection process. Moreover, dicistronic expression vectors tend to undergo deletion or rearrangement under amplification conditions, in an uncontrolled manner, increasing the chances that amplified cells will no longer express the protein of interest. Internal ribosome entry sites (IRES) are a type of regulatory element found in several viruses and cellular RNAs (reviewed in McBratney et. al. Current Opinion in Cell Biology 5:961, 1993). IRES increase the efficiency of translation of the selectable marker gene, and are thus useful in enhancing both the selection and amplification process (Kaufman R. J., et al., Nucleic Acids Res. 19:4485, 1991). Nonetheless, the available evidence indicates that dicistronic mRNAs accumulate to lower levels than monocistronic mRNAs, possibly because of reduced mRNA stability of the longer message.
Because the amount of recombinant protein produced by a transfected cell is generally proportional to the amount of mRNA available for translation of the protein, the use of dicistronic expression vectors may result in low levels of production of the desired recombinant protein. Accordingly, there is a need in the art to develop improved methods that retain the utility of a selectable, amplifiable marker such as DHFR, while increasing the proportion of mRNAs encoding the desired recombinant protein. Moreover, there is a need to develop methods that facilitate selection of those transfectants that integrate into more transcriptionally active sites, and that allow production of useful levels of recombinant protein from mammalian cells in a relatively short period of time.
In one embodiment of the invention, an expression vector comprises a DNA encoding a first protein, operably linked to a DNA encoding a second protein, wherein a DNA encoding a polyadenylation (polyA) site is inserted between the DNA encoding the first protein of interest and the DNA encoding the second protein, such that the DNA encoding the internal polyadenylation site is operably linked to the DNA encoding the first. A preferred second protein is selectable marker, preferably dihydrofolate reductase (DHFR); other amplifiable markers are also suitable for use in the inventive expression vectors.
Preferably, the polyadenylation signal utilized to provide the internal polyadenylation site is an SV40 polyadenylation signal, more preferably, the late SV40 polyadenylation signal, and most preferably, a mutant version of the late SV40 polyadenylation signal. The preferred polyadenylation signals are presented in the Sequence Listing and described further below. In another embodiment of the invention, the polyadenylation signal is inducible.
The expression vector may further comprise an IRES sequence between the DNA encoding the first protein, and the DNA encoding the second protein, operably linked to both and downstream of the internal polyadenylation site. Alternatively, the expression vector may comprise mRNA splice donor and acceptor sites substantially as described by Lucas et al. infra.
Another aspect of the invention comprises an expression vector into which a DNA encoding a protein. Such an expression vector comprises a site into which a DNA encoding a recombinant, heterologous protein can be inserted (referred to as a cloning site), such that it is operably linked to an internal polyadenylation site and a DNA encoding a second protein (such as a selectable marker). Optionally, other regulatory elements may also be included, for example, an IRES sequence downstream of the internal polyadenylation site, or mRNA splice donor and acceptor sites substantially as described by Lucas et al. infra, operably linked to the internal polyadenylation site and the DNA encoding the second protein. An expression-augmenting sequence element (EASE) may also be included upstream of the cloning site, operably linked thereto.
Host cells can be transfected with the inventive expression vectors, yielding stable pools of transfected cells. Accordingly, another embodiment of the invention provides a transfected host cell; yet another embodiment provides a stable pools of cells transfected with the inventive expression vector. Also provided are cell lines cloned from pools of transfected cells. Preferred host cells are mammalian cells. In a most preferred embodiment, the host cells are CHO cells.
The invention also provides a method for obtaining a recombinant protein, comprising transfecting a host cell with an inventive expression vector, culturing the transfected host cell under conditions promoting expression of the protein, and recovering the protein. In a preferred application of this invention, transfected host cell lines are selected with two selection steps, the first to select for cells expressing the dominant amplifiable marker, and the second step for high expression levels and/or amplification of the marker gene as well as the gene of interest. In a most preferred embodiment, the selection or amplification agent is methotrexate, an inhibitor of DHFR that has been shown to cause amplification of endogenous DHFR genes and transfected DHFR sequences.